The enzyme 3-oxo-delta5-steroid isomerase (KSI) is a key enzyme in the biosynthetic pathway for steroid hormones. the importance of steroid hormone deprivation protocols in current cancer therapy makes a detailed understanding of the mechanisms of the enzymes involved in this pathway of major importance. Ultimately, it is hoped that a selective inhibition of one of these enzymes will allow a pharmacological approach to hormone- dependent cancers to be achieved, surpassing current surgical methodology. This proposal for supplemental funding describes plans to investigate the 3-dimensional structure of the KSI from Pseudomonas testosteroni and its D38E mutant. These studies are required to enable us to correlate our kinetic and mechanistic results with the structure of the enzyme. Two- dimensional and three-dimensional NMR investigations will be carried out on 500 MHz and 600 MHz spectrometers and the results will be analyzed by NMR- based distance geometry methodologies. NMR studies will be extended to protein-inhibitor complexes to gain additional insights into structural aspects of catalysis. Preliminary data presented here show that this approach is feasible for the structural determination of KSI.